Project Summary The most promising therapeutic approach to cure HIV infection is the ?activate-and-kill? strategy in which proteins or small molecules, called latency reversing agents (LRAs), are used to activate latent viruses and then infected cells are killed due to viral cytopathic effects or by the host immune response. There are several limitations to this approach, including incomplete viral reactivation and cell death, and the fact that LRAs are administered in a non-specific manner because there are no known biomarkers that distinguish latent HIV- infected CD4+ T cells from uninfected cells. Previous attempts to discover biomarkers that distinguish latently infected cells from uninfected cells were conducted in the absence of stimulation and the differences measured were modest. Stimulating signaling networks and following the dynamics of protein activation over time has proved in other contexts to be a successful way to differentiate between healthy and diseased cells. Here we propose to use a multivariate systems approach to explore if latent HIV infection alters signaling protein interactions, and to determine if these altered interactions are promising therapeutic targets. The overall objective of this proposal is to identify novel signaling targets that can be perturbed in combination with LRAs to specifically enhance viral reactivation and/or cell death in latently infected cells, while minimizing responses in uninfected cells. The central hypothesis, supported by preliminary data, is that signaling network regulation of T cell activation and cell death is altered in latent HIV-infected CD4+ T cells as compared to uninfected cells. To identify network-level changes in signaling interactions in latent HIV-infected cells, we will use computational analyses of measurements of signaling proteins, T cell activation markers, and viral and cell death proteins collected by CyTOF (cytometry by time-of-flight) following stimulation. In Aim 1, CyTOF will be used to identify the network of signaling pathways that are altered by latent HIV infection following TCR stimulation and stimulation with other drugs. In Aim 2, we will use systems analysis of the single-cell CyTOF data sets to predict signaling targets that will uniquely perturb responses in latently infected cells, and then validate these targets experimentally to demonstrate that viral reactivation and/or cell death can be synergistically increased in latently infected cells without affecting uninfected cells. This contribution will be significant because it is expected to ultimately improve the design of therapeutic combinations of LRAs and administration schedules to more effectively and specifically target the latently infected CD4+ T cell reservoir in patients to reactivate virus and induce cell death. The proposed research is innovative because it represents a substantive departure from earlier efforts by using a systems analyses of multivariate single-cell measurements of signaling following T cell stimulation to discover altered signaling networks in primary latently infected T cells. PHS 398/2590 (Rev. 06/09) Continuation Format Page